The present invention is in the field of dehydrogenases that are related to the retinol dehydrogenase subfamily, recombinant DNA molecules and protein production. The present invention specifically provides novel dehydrogenase polypeptides and proteins and nucleic acid molecules encoding such peptide and protein molecules, all of which are useful in the development of human therapeutics and diagnostic compositions and methods.
Dehydrogenases, particularly members of the retinol dehydrogenase subfamily, are a major target for drug action and development. Accordingly, it is valuable to the field of pharmaceutical development to identify and characterize previously unknown members of these subfamily of dehydrogenases. The present invention advances the state of the art by providing a previously unidentified human dehydrogenases that have homology to members of the retinol dehydrogenase subfamilies.
Retinol Dehydrogenase
The human protein, and encoding gene, provided by the present invention is related to the dehydrogenase protein family, and shows a particularly high degree of similarity to retinol dehydrogenases. Retinol dehydrogenase catalyzes the primary and rate-limiting step in the synthesis of retinoic acid from retinol. Retinoic acid controls a wide variety of important biological systems by regulating eukaryotic gene expression (Chai et al., J Biol Chem Feb. 24, 1995;270(8):3900-4). Retinoic acid is a powerful regulator of gene expression; novel retinol dehydrogenases are useful for modulating the synthesis of retinoic acid and, therefore, are useful for modulating gene expression in numerous important biological systems. Furthermore, retinol dehydrogenase 4 is useful for catalyzing the synthesis of not only retinoic acid, but also dihydrotestosterone from 3alpha-androstane-diol (Gough et al., J Biol Chem Jul. 31, 1998;273(31):19778-85). Like retinoic acid, dihydrotestosterone is also a powerful modulator of gene expression. Retinol dehydrogenase 4 also oxidizes all-trans-retinol and 13-cis-retinol to corresponding aldehydes and oxidizes the 3alpha-hydroxysteroids androstane-diol and androsterone to dihydrotestosterone and androstanedione, respectively (Gough et al., J Biol Chem Jul. 31, 1998;273(31):19778-85).
Retinoic acid is the active form of vitamin A, which is necessary for the normal growth and differentiation of the epidermis. Retinaldehyde is an intermediate in the synthesis of retinoic acid from retinol in epidermal keratinocytes. Retinol dehydrogenase catalyzes the initial and rate-limiting step that generates retinaldehyde from retinol (Jurukovski et al., Mol Genet Metab May 1999;67(1):62-73). Therefore, novel retinol dehydrogenases are useful for modulating the growth and differentiation of the epidermis, such as for treating epidermal/skin disorders or enhancing skin health and appearance.
Vitamin A is a pigment essential to vision. Vitamin A comes from the enzymatic conversion of carotenoids, yellow pigments common to carrots and other vegetables, to retinol. Deficiency of vitamin A and insufficient retinol production leads to a variety of maladies in humans and experimental animals. Symptoms of deficiency include vision related disorders such as xerophthalmia and night blindness; dry skin and dry mucous membranes; retarded development and growth; and sterility in male animals.
Cleavage of beta-carotene yields two molecules of retinol; oxidation of retinol forms retinal. Retinal and opsin combine to produce rhodopsin, a visual pigment found in nature. The excitation of rhodopsin with visible light triggers a series of photochemical reactions and conformational changes in the molecule which result in the electrical signal to the brain that are the basis of visual transduction (Lehninger et al. (1993) Principles of Biochemistry, Worth Publishers, New York, N.Y.).
Retinol dehydrogenase (RoDH) catalyzes the conversion of retinol to retinal; retinal dehydrogenase converts retinal to retinoate. Retinoate is a retinoid and a hormone that controls numerous biological processes by regulating eukaryotic gene expression. Retinoids, like steroid and thyroid hormones, diffuse directly across the plasma membrane and bind to intracellular receptor proteins. Binding activates the receptors that interact with signaling pathways (Vettermann et al. (1997) Mol. Carcinog. 20: 58-67), and regulate the transcription of specific genes, particularly those mediating vertebrate development (Alberts et al. (1994) Molecular Biology of the Cell, Garland Publishing, Inc., New York, N.Y.). Retinol is known to be important in epithelial development (Haselbeck et al. (1997) Dev. Dyn. 208: 447-453; and Attar et al. (1997) Mol. Endocrinol. 11: 792-800) and in the development of the central nervous system (Maden et al. (1997) Development 124: 2799-2805). In Maden""s studies on quail embryos, absence of vitamin A, lead to severe deficits including lack of a posterior hindbrain. Conversely, injection of retinol before gastrulation of the embryo prevented positional apoptosis and corrected the CNS defects.
The universal chromophore of visual pigments is 11-cis retinaldehyde, which is generated by 11-cis retinol dehydrogenase, a membrane-bound enzyme abundantly expressed in the retinal pigment epithelium of the eye. The gene that encodes 11-cis retinol dehydrogenase may be involved in hereditary eye diseases (Simon et al. (1996) Genomics 36: 424-430).
Chai et al. have identified, cloned, and expressed two isoforms of retinol dehydrogenase, RoDH(I) and (RoDH(II) (1995, J. Biol. Chem. 270: 28408-28412). The deduced amino acid sequence shows that RoDH(I) and RoDH(II) are short-chain dehydrogenases/reductases that share 82% identity. Retinol is the substrate for RoDH(II) which has a higher affinity for NADP than NAD and is stimulated by ethanol and phosphatidyl choline. Although RoDH(II) is not inhibited by the medium-chain alcohol dehydrogenase inhibitor, 4-methylpyrazole, it is inhibited by phenylarsine oxide and carbenoxolone. Chai et al. reported detection of RoDH(I) and RoDH(II) mRNA in rat liver, but RNase protection assays revealed RoDH(I) and RoHD(II) mRNA in kidney, lung, testis, and brain. Based on these data, Chai et al. concluded that RoDH has tissue specific expression.
The retinol signaling pathway plays an important role in human disorders and diseases. Retinoic acid receptors (RARs; -alpha, -beta, and -gamma) are retinoid-activated transcription factors, which mediate effects of retinoids on gene expression. Alterations in receptor expression or function could interfere with the retinoid signaling pathway. Interference with the pathway may enhance cancer development. Vitamin A analogs (retinoids) which interact with RARs, suppress oral and lung carcinogenesis in animal models and prevent the development of tumors in head, neck, and lung cancer patients (Lotan R. 1997 Environ. Heath Perspect. 105 Suppl. 4: 985-988). Lotan reported that RAR beta expression is lost at early stages of carcinogenesis in the aerodigestive tract.
Retinol dehydrogenase is implicated in embryonic development. The studies of Maden et al. (supra) suggest that retinol may play a significant role in controlling apoptosis during development of the central nervous system. Retinoids are also implicated in epidermal development. Attar et al. (1 997, Mol. Endocrinol. 11: 792-800) showed that disruption of epidermal barrier function results in extremely high incidences of neonatal mortality in pups.
Retinoic acid acts as an important signal during development, particularly in the development of the neural axis, and defects in retinoic acid synthesis due to defects in retinaldehyde dehydrogenase proteins or the encoding genes can severely affect development and lead to early embryonic death. For example, targeted disruption of the retinaldehyde dehydrogenase 2 gene in the mouse has been shown to cause embryonic death at midgestatation. Furthermore, such mutated embryos lack axial rotation, exhibit shortening along the anterioposterior axis and have an open neural tube, and lack limb buds. Additional severe defects were observed in the hearts, frontonasal regions, and otocysts of these mutated embryos. These defects were caused by a blockage of embryonic retinoic acid synthesis. These experiments established that retinoic acid synthesized by the postimplantation embryo is a critical developmental hormone and that a lack of retinoic acid causes early embryonic death (Niederreither et al., Nature Genet. 21: 444-448,1999).
In addition, retinol dehydrogenase activity is linked to hereditary eye diseases (Simon et al. (1996) Genomics 36: 424-430). Autosomal recessive childhood-onset severe retinal dystrophy (arCSRD) is a heterogeneous group of disorders that affect rod and cone photoreceptors simultaneously. Disease genes implicated in arCSRD are expected to encode proteins present in the neuroretina or in the retinal pigment epithelium (RPE). RPE65, a tissue-specific and evolutionarily highly conserved 61 kD protein, is the first disease gene in this group of inherited disorders that is expressed exclusively in RPE, and may play a role in vitamin A metabolism of the retina (Gu et al. (1997) Nat. Genet. 17: 194-197).
Pityriasis rubra pilaris (PRP) is an idiopathic erythematous scaling eruption which can be difficult to distinguish from psoriasis. The expression of RoDH(II) in the retinol signaling pathway may be of pathogenetic importance in the diagnosis of PRP (Magro, C. M. and Crowson, A. N. (1997) J. Cutan. Pathol. 24: 416-424).
For a further review of dehydrogenases, particularly retinol dehydrogenases, see Chai et al., Gene Mar. 9, 1996;169(2):219-22; Chai et al., J Biol Chem Dec. 26, 1997;272(52):33125-31; Gough et al., J Biol Chem Jul. 31, 1998;273(31):19778-85; Ono et al., Molec. Cell. Biol. 18: 6939-6950, 1998; Wang et al., J. Biol. Chem. 271: 16288-16293, 1996; and Zhao et al., Europ. J. Biochem. 240: 15-22, 1996.
The discovery of new human dehydrogenase proteins, particularly members of the retinol dehydrogenase subfamily, and the polynucleotides encoding these proteins satisfies a need in the art by providing new compositions which are useful in the diagnosis, prevention and treatment of biological processes associated with human. diseases.
The present invention is based in part on the identification of amino acid sequences of human dehydrogenase polypeptides and proteins that are related to the retinol dehydrogenase subfamily, as well as allelic variants and other mammalian orthologs thereof. These unique peptide sequences, and nucleic acid sequences that encode these peptides, can be used as models for the development of human therapeutic targets, aid in the identification of therapeutic proteins, and serve as targets for the development of human therapeutic agents that modulate dehydrogenase activity in cells and tissues that express the dehydrogenase. The protein of the present invention is expressed in human placenta, as indicated by the library source of the cDNA clone (indicated in FIG. 1).